Instrumentation Design of a High-Speed Fluorescence Lifetime Imaging Microscope Tailored to High-Throughput Screening for Drug Discovery

Fluorescence lifetime imaging microscopy (FLIM) is a powerful imaging modality that can provide unique information related to the biological microenvironment of a sample.  For instance, when applied to quantifying Förster resonant energy transfer (FRET), the decay characteristics of the fluorescence can be indicative of the degree of protein-protein interaction occurring at a subcellular level.  There is a clear interest at the drug discovery level to harness the potential of FLIM-FRET and other FLIM techniques to better characterize and understand the mechanisms of their drug leads.

To properly test and isolate the effectiveness of drug leads under varied conditions and doses, the drug discovery process uses high-content screening (HCS) microscopes to image 100,000 samples per day. FLIM techniques have so far been unable to provide a suitable combination of acquisition speed and resolution to fill this demand. Widefield FLIM techniques suffer from out-of-focus light, which is especially detrimental to fitting the lifetimes of fluorescence decays. Typical raster-scanning confocal approaches can offer excellent spatial and temporal resolution with a photomultiplier tube as a single-point detector, but are inherently slow to collect a full FLIM image.

We are designing a system to meet these HCS requirements by combining a multiplexed raster scanning approach with a streak camera and high-speed readout camera. In a streak camera, photons incident on the entrance slit are focused to a photocathode. The photoelectrons generated at the photocathode are accelerated through a vacuum tube, across a perpendicular time-varying electric field, which can deflect the electrons to the left or right based on their arrival time. The resulting readout would consist of a spatial axis and a temporal axis. The slit of a streak camera is an excellent method of multiplexing the fluorescence lifetime capture along its spatial axis. Our streak camera (Optronis SC-10) can be filled with up to 100 resolvable optical fiber channels.

A lenslet array is used to generate 10x10 focal points on the sample, which can then be scanned over the sample with an x-y galvo system. Given sufficient excitation power, this offers a 100x improvement on acquisition speed.  The galvo scanner also provides a descan of the fluorescent signals on the return path, such that the fluorescence signals can be focused into a fixed 2-dimensional optical fiber array. To accommodate the streak camera slit, the 100 fiber channels are rearranged from 10x10 to 1x100.

We will discuss the current performance of the system, and the potential improvements for each component to get closer to the required acquisition speed. In particular, there is interest in investigating the use of solid-state devices to replace the streak camera. Single-photon avalanche diode (SPAD) arrays are an emerging technology that could greatly simplify the instrumentation, reduce the acquisition time, and improve the light collection efficiency. There are also potential improvements in frame rate and light economy concerning our excitation sources, optics and readout camera.